Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence.

Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.

The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells.

Streptococcus mutans is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (c, e, f, and k), with serotype c strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes e and f are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the Galleria mellonella (greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene, cnm, which was previously shown to bind to collagen and laminin in vitro. Inactivation of cnm rendered the organisms unable to invade HCAEC and attenuated their virulence in G. mellonella. Notably, the cnm knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective cnm mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for S. mutans invasion of endothelial cells and possibly represents an important virulence factor of S. mutans that may contribute to cardiovascular infections and pathologies.

Peroxisome proliferator-activated receptor gamma ligands enhance human B cell antibody production and differentiation.

Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)gamma/RXRalpha pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARgamma. We also show that nanomolar levels of natural (15-deoxy-Delta(12,14)-prostaglandin J(2)) or synthetic (rosiglitazone) PPARgamma ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARgamma antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARgamma and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRalpha ligand (9-cis-retinoic acid) and PPARgamma ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARgamma ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARgamma ligands. These new findings indicate that low doses of PPARgamma/RXRalpha ligands could be used as a new type of adjuvant to stimulate Ab production.

A review of mentoring deaf and hard-of-hearing scholars.

Diversification of the scientific workforce usually focuses on recruitment and retention of women and underrepresented racial and ethnic minorities but often overlooks deaf and hard-of hearing (D/HH) persons. Usually classified as a disability group, such persons are often members of their own sociocultural linguistic minority and deserve unique support. For them, access to technical and social information is often hindered by communication- and/or language-centered barriers, but securing and using communication access services is just a start. Critical aspects of training D/HH scientists as part of a diversified workforce necessitates: (a) educating hearing persons in cross-cultural dynamics pertaining to deafness, sign language, and Deaf culture; (b) ensuring access to formal and incidental information to support development of professional soft skills; and (c) understanding that institutional infrastructure change may be necessary to ensure success. Mentorship and training programs that implement these criteria are now creating a new generation of D/HH scientists.

The VE-cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells.

Fibrin deposition and exudation of plasma fibrinogen (Fg) have long been recognized as hallmarks of inflammation, cardiovascular disease and neoplasia. The Fg-beta(15-42) domain binds to the endothelial cell adhesion molecule, VE-cadherin, promoting endothelial cell proliferation, angiogenesis and leukocyte diapedesis. Furthermore, spontaneous blood-borne and lymphatic metastasis of some types of tumor emboli requires plasma fibrin(ogen); however, the molecular mechanisms by which this occurs are poorly understood. We sought to determine whether Fg-beta(15-42) and VE-cadherin binding interactions promote endothelial barrier permeability and breast cancer cell transendothelial migration (TEM) using transwell insert culture systems. Synthetic peptides containing/missing residues beta(15-17) critical for Fg-beta(15-42) binding to VE-cadherin, and antibodies that bind to Fg-beta(15-21) (T2G1) and VE-cadherin (BV9) were used to induce or inhibit Fg-mediated permeability and TEM. Fg induced dose-dependent permeability of human umbilical vein and microvascular endothelial but not epithelial cell barriers. Maximal Fg-induced endothelial permeability required Fg-beta(15-42) and VE-cadherin-binding interactions involving Fg-beta(15-17). Fg-induced TEM of malignant MDA-MB-231 and MCF-7 breast cancer cells also required Fg-beta(15-42) and VE-cadherin binding; however, such TEM was independent of E-cadherin or estrogen receptor expression. In contrast, Fg did not induce TEM of nonmalignant MCF-10A breast epithelial cells. Fg-induced endothelial permeability was retained in the presence of MDA-MB-231 but inhibited in the presence of MCF-10A cells. It is intriguing to speculate that loss of Fg-beta(15-42) binding by premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype. Hence, Fg-beta(15-42) represents a potential molecular target for therapeutic intervention of breast cancer metastasis.

Primary human endothelial cells support direct but not antibody-dependent enhancement of dengue viral infection.

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2-16681 virus. Viral replication was detected as early as 24 hr post-infection by RT-PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen-positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcgammaRI and FcgammaRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock-infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re-examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome.

Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation.

Hepatic fibrinogen (FBG) is upregulated during an acute phase response (APR) induced by glucocorticoids and interleukin (IL)-6. Furthermore, intestine and lung epithelium synthesize FBG after exposure to inflammatory mediators, and both plasma and lung cell-derived FBG, along with fibronectin, assemble in detergent-insoluble extracellular matrices (ECM) of pneumocytes and fibroblasts independent of thrombin or plasmin cleavage. An epitope cryptic in soluble FBG (beta(15-21)) but exposed in matrix-FBG and fibrin induces cell proliferation and actin cytoskeleton reorganization during wound repair and angiogenesis. Although fibrin(ogen) is involved in hemostasis and homeostasis, mechanisms regulating extrahepatic FBG expression remain unexplored. Herein we examined FBG production by lung compared to liver epithelial cell lines in response to dexamethasone (DEX)+IL-6. Regulated synthesis of HepG2-FBG follows the pathway shown for constitutive synthesis by liver epithelium. Constitutive A549-FBG expression was not detectable, however, intracellular FBG precursors in DEX+IL-6-treated A549 lung cells were similar to HepG2 cells with two notable exceptions. The relative rate of chain synthesis in HepG2 cells was unequal, whereas nascent synthesis of all three chains occurred at equivalent rates in stimulated A549 cells. Unlike HepG2 cells, which rapidly secreted intact FBG, nascent dimeric FBG accumulated in the A549 cell-associated fraction prior to release into medium. Furthermore, soluble A549-FBG was susceptible to thrombin and plasmin cleavage. Interestingly, many functionally diverse proteins possess FBG-related domains that direct cell-fate determination during development or wound repair, suggesting that extrahepatic FBG biosynthesis evoked only during inflammation plays such a role during localized injury and repair to restore tissue homeostasis.

A putative role for platelet-derived PPARγ in vascular homeostasis demonstrated by anti-PPARγ induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis.

Widely known for its role in adipogenesis and energy metabolism, PPARγ also plays a role in platelet function. To further understand functions of platelet-derived PPARγ, we produced rabbit polyclonal (PoAbs) and mouse monoclonal (MoAbs) antibodies against PPARγ 14mer/19mer peptide-immunogens. Unexpectedly, our work produced two key findings. First, MoAbs but not PoAbs produced against PPARγ peptide-immunogens displayed antigenic crossreactivity with highly conserved PPARα and PPARß/δ. Similarly, Santa Cruz PoAb sc-7196 was monospecific for PPARγ while MoAb sc-7273 crossreacted with PPARα and PPARß/δ. Second, immunized rabbits and mice exhibited unusual pathology including cachexia, excessive bleeding, and low platelet counts leading to thrombocytopenia. Spleens from immunized mice were fatty, hemorrhagic and friable. Although passive administration of anti-PPARγ PoAbs failed to induce experimental thrombocytopenia, megakaryocytopoiesis was induced 4-8-fold in mouse spleens. Similarly, marrow megakaryocytopoiesis was enhanced 1.8-4-fold in immunized rabbits. These peptide-immunogens are 100% conserved in human, rabbit and mouse; thus, immune-mediated platelet destruction via crossreactivity with platelet-derived PPARγ likely caused bleeding, thrombocytopenia, and compensatory megakaryocytopoiesis. Such overt pathology would cause significant problems for large-scale production of anti-PPARγ PoAbs. Furthermore, a major pitfall associated with MoAb production against closely related molecules is that monoclonicity does not guarantee monospecificity, an issue worth further scientific scrutiny.

Role of peroxisome proliferator-activated receptor gamma and its ligands in the treatment of hematological malignancies.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a multifunctional transcription factor with important regulatory roles in inflammation, cellular growth, differentiation, and apoptosis. PPARgamma is expressed in a variety of immune cells as well as in numerous leukemias and lymphomas. Here, we review recent studies that provide new insights into the mechanisms by which PPARgamma ligands influence hematological malignant cell growth, differentiation, and survival. Understanding the diverse properties of PPARgamma ligands is crucial for the development of new therapeutic approaches for hematological malignancies.

Cell type-specific differential induction of the human gamma-fibrinogen promoter by interleukin-6.

During an acute phase response, interleukin-6 (IL-6) and glucocorticoids up-regulate expression of the three fibrinogen (FBG) genes (fga, fgb, and fgg) in liver and lung epithelium; however, little constitutive lung expression occurs. Recently, we showed that the magnitude of Stat3 binding to three IL-6 motifs on the human gammaFBG promoter correlates negatively with their functional activity in hepatocytes, although these cis-elements are critical for promoter activity. We determined the role of IL-6-receptor-gp130-Stat3 signaling in IL-6 activation of the gammaFBG promoter in liver and lung epithelial cells. Although IL-6 induced gammaFBG promoter activity approximately 30-fold in HepG2 cells, it was increased only 2-fold in lung A549 cells. Equivalent production of gp130 was demonstrated in both cell types by Western blotting; however, lower production of both IL-6-receptor and Stat3 explains, in part, reduced activity of the gammaFBG promoter in lung cells. Dexamethasone potentiated IL-6 induction of the gammaFBG promoter 2.3-fold in both HepG2 and A549 cells for a combined increase in promoter activity of 70-fold or 4.5-fold, respectively. Dexamethasone potentiation is likely due to the induction of IL-6-receptor expression as well as prolonged intensity and duration of Stat3 activation. By circumventing IL-6-receptor-gp130-coupled signaling with ectopic expression of the granulocyte colony-stimulating factor receptor (GCSFR)-gp130(133) chimeric receptor, overexpression of Stat3 induced gammaFBG promoter activity 30-fold in A549 cells. Together, the data suggest tissue-specific differences in IL-6-receptor-gp130-coupled signaling, thereby limiting the extent of Stat3 activation and gammaFBG expression during lung inflammation.

Functional analysis of interleukin 6 response elements (IL-6REs) on the human gamma-fibrinogen promoter: binding of hepatic Stat3 correlates negatively with transactivation potential of type II IL-6REs.

Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately up-regulated by IL-6 and glucocorticoids. Except for rat gammaFBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human gammaFBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to gammaFBG IL-6REs was confirmed by cross-competition with alpha2-macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at -306 to -301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of gammaFBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced gammaFBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human gammaFBG IL-6REs was low compared with Stat3 binding to the alpha2-macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR.

Epitope mapping of a protective monoclonal antibody against Pneumocystis carinii with shared reactivity to Streptococcus pneumoniae surface antigen PspA.

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the M(r) of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.

Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration.

Fibrinogen (FBG) assembles into matrix fibrils of fibroblasts, lung and mammary epithelial cells, but not endothelial cells. Furthermore, cryptic beta15-21 residues are exposed in FBG fibrils with no evidence of thrombin or plasmin proteolysis. Herein, the effects of FBG on migration and proliferation of wounded dermal fibroblasts were investigated. FBG preassembled into matrix prior to scrape-wounding induced 3H-thymidine incorporation 8-fold and shortened the time to wound closure 1.6-fold +/- 0.1-fold. FBG added immediately after wounding did not enhance either response. Fibroblast growth factor-2/platelet-derived growth factor (FGF-2/PDGF) stimulated cell proliferation 2.2-fold for FGF-2 and 3.2-fold for PDGF and wound closure 1.5-fold +/- 0.1-fold in the absence of matrix-FBG. Surprisingly, exogenous growth factors had negligible effect on wound closure and cell proliferation already enhanced by matrix-FBG. Matrix-FBG-enhanced wound closure required active assembly of an FBG-fibronectin matrix, engagement of alphavbeta3, and FBG Aalpha-RGDS572-575 integrin recognition sites; Aalpha-RGDF95-98 sites were not sufficient for matrix-FBG assembly, enhanced wound closure, or cell proliferation. Although Bbeta1-42 was not necessary for matrix assembly, it was required for matrix-FBG-enhanced cell migration. These data indicate that FBG serves as an important matrix constituent in the absence of fibrin formation to enhance wound repair and implicate Bbeta1-42 as a physiologic inducer of signal transduction to promote an intermediate state of cell adhesion and a migratory cell phenotype.

The incorporation of fibrinogen into extracellular matrix is dependent on active assembly of a fibronectin matrix.

Fibrinogen is a soluble protein produced by hepatocytes and secreted into plasma, where it functions in hemostasis. During inflammation, the hepatic synthesis of fibrinogen is induced 2-10 fold. Recent studies demonstrate that after an inflammatory stimulus, fibrinogen gene expression and protein production is upregulated in lung epithelial cells, where it is secreted basolaterally and consequently deposited into the extracellular matrix in fibrils that extensively colocalize with fibronectin fibrils. In this study, we show that the deposition of fibrinogen into the matrix of fibroblasts occurred rapidly and in a Rho-dependent manner in response to serum or lysophosphatidic acid; RhoA GTPase signaling is also required for fibronectin matrix assembly. Using mouse embryonic fibronectin-null cells, we show that incorporation of exogenous fibrinogen into matrix fibrils occurred only in the presence of exogenous fibronectin, which is also assembled into matrix fibrils. Furthermore, treatment of fibroblasts and fibronectin-null cells with an antibody that inhibits fibronectin matrix assembly impaired incorporation of fibrinogen into matrix fibrils. Collectively, these data suggest that incorporation of fibrinogen into the extracellular matrix requires active fibronectin polymer elongation into matrix fibrils. From these data, we hypothesize that fibrinogen deposition rapidly changes the topology of the extracellular matrix to provide a surface for cell migration and matrix remodeling during tissue repair.